Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.
Methods for the chemical synthesis of isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A. Chem. 220:215-220, 2004). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly. The production of isobutanol from plant-derived raw materials would minimize greenhouse gas emissions and would represent an advance in the art.
Isobutanol is produced biologically as a by-product of yeast fermentation or by recombinantly engineered microorganisms modified to express a butanol biosynthetic pathway for producing butanol. See e.g., U.S. Pat. No. 7,851,188, which is incorporated herein by reference in its entirety. As a component of “fusel oil” that forms as a result of the incomplete metabolism of amino acids by fungi, isobutanol is specifically produced from catabolism of L-valine. After the amine group of L-valine is harvested as a nitrogen source, the resulting α-keto acid is decarboxylated and reduced to isobutanol by enzymes of the so-called Ehrlich pathway (Dickinson et al., J. Biol. Chem. 273:25752-25756, 1998).
One of the key yield loss mechanisms in yeast butanol production is the loss of carbon and reducing equivalents that are diverted from glycolysis by the conversion of dihydroxyacetone phosphate to glycerol. The first step in this conversion is catalyzed by an enzyme called glycerol-3-phosphate dehydrogenase (GPD). Eliminating GPD, and therefore glycerol production, in butanol-producing yeast, has been proposed previously. However, glycerol is required for growth and is an osmoprotectant.
Accordingly, methods of increasing butanol yield and decreasing glycerol production represent an advance in the art.